A variety of cancer-critical signaling pathways require recruitment of FYVE, PX and ENTH domain-containing proteins to phosphoinositide (Pl)-enriched cell membranes. Many of these proteins are directly implicated in tumorigenesis and/or involved in down-regulation of proliferative pathways by internalizing oncogenic growth factor receptors (GFRs). The recent advances in understanding of the fundamental role of endocytosis of activated GFRs and the components of endocytic machinery in tumor development and progression suggest that the PI-binding proteins could be important pharmacological targets in cancer treatment. Despite the critical role of the PI-recognizing domains in human physiology and cancer, our knowledge of how these proteins are activated and recruited to endocytic membranes remains very limited. In the proposed research a set of biochemical, biophysical and structural biology tools will be used to elucidate the precise molecular mechanisms underlying the membrane targeting and function of the FYVE, PX and ENTH domains. In particular, the functional importance of the PI's headgroup recognition, hydrophobic insertion, non-specific electrostatic interactions and pH of media will be established. The hypothesis to be tested is whether the membrane lipid composition and acidic lumenal or cytosolic environment regulate the membrane targeting and function of the PI-binding domains. The three specific aims are proposed. The first objective is to characterize the effect of insertion on membrane recruitment of the PI-binding domains. The second goal is to determine how the lipid composition of membranes affects membrane targeting. In the third aim, the effect of the acidic environment on membrane targeting by the PI-binding proteins will be established. This research will offer comprehensive understanding of how the PI-binding domains recognize PIs and interact with lipid bilayers at the atomic level. Elucidating the molecular mechanisms of membrane targeting and activation of the PI-binding proteins will help us to find ways to specifically control and inhibit the oncogene receptor signaling that leads to the growth and division of cancer cells. These studies will aid in deeper understanding of how the lipid signals are recognized and will help to identify new prognostic and diagnostic markers and targets for cancer therapy. The results generated by this research may provide new approaches to rational design of anti-tumor therapeutic agents and systems for delivering drugs into cancer cells.